Amphipathic coating for modulating cellular adhesion composition and methods

ABSTRACT

The present invention provides an anti-thrombogenic and cellular-adhesion coating composition for blood-contacting surfaces. The coating comprises a covalent complex of from 1 to 30 hydrophobic silyl moieties of Formula I:                    
     wherein R 1  is an C 1-18  alkyl or C 6-32  aryl group, each R 2  is independently selected from the group consisting of C 1-18  alkyl and C 6-32  aryl, R 3  is N or O, n is a number from 1 to 10, directly bound to a heparin molecule via covalent bonding, with an adhesive molecule directly bound to the heparin molecule. In one embodiment, the coating comprises benzyl-(1,2 dimethyl)disilyl heparin, wherein an adhesive molecule, such as fibronectin, is bound to the heparin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 09/399,119, entitled Non-Thrombogenic CoatingCompositions and Methods for Using Same, to Ray Tsang and ShigemasaOsaki, filed on Sep. 20, 1999, and now abandoned, which is acontinuation patent application of 09/159,276, field on Sep. 22, 1998,and now U.S. Pat. No. 5,955,588, entitled Non-Thrombogenic CoatingCompositions and Methods for Using Same, to Ray Tsang and ShigemasaOsaki, and the specification of each of the foregoing is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention (Technical Field)

The present invention relates to coatings and methods of use ofnon-thrombogenic compositions for selectively inhibiting and promotingcellular attachment, including a silyl-heparin-fibronection compositionfor promoting cellular attachment.

2. Background Art

Note that the following discussion refers to a number of publications byauthor(s) and year of publication, and that due to recent publicationdates certain publications are not to be considered as prior artvis-a-vis the present invention. Discussion of such publications hereinis given for more complete background and is not to be construed as anadmission that such publications are prior art for patentabilitydetermination purposes.

Heparin is naturally present in various tissues, including liver andlung, as well as the luminal surface of endothelial cells. It iscomposed of repeating units of D-glucuronic acid and D-glucosamine, bothsulfated, in a 1,4-α linkage. Heparin is an anticoagulant, and it hasbeen reported that on the surface of endothelial cells heparin minimizesfibrin accumulation. When administered as a parenteral drug, heparinactivates anti-thrombin III, which leads to inactivation of thrombin andultimately systemic inhibition of fibrin formation.

A number of medical devices that come in contact with blood have beencoated with heparin with the goal of taking advantage of itsthrombo-resistant nature. Stents, catheters, oxygenator fibers, andcardiac bypass circuits are examples of medical devices that have beencoated with heparin (Niimi et al., Anesth Analg 89:573-9, 1999; lnui etal., Artif Organs, 23:1107-12, 1999). Various strategies have beendeveloped to attach heparin to medical polymer surfaces includingchemical conjugation (Siefert et al., J Biomater Sci Polym Ed, 7:277-87,1995), plasma glow discharge methods (Kim et al., Biomaterials,21:121-30, 2000), the combination of both, and hydrophobic interactionas described herein (U.S. Pat. No. 5,955,588).

Heparin has a number of other biological actions related to its presencein the extracellular matrix. In the extracellular matrix, heparin andits chemical relative heparan sulfate is complexed into a scaffoldingonto which cells attach. In this scaffolding, heparin is bound byfibronectin and other adhesive molecules, which in turn bind to cells.Extracellular matrix heparin and heparin sulfate also act as reservoirsfor growth factors, not only binding growth factors but also protectingthem from protease degradation. Fibroblast growth factor (FGF),platelet-derived growth factor (PDGF), and bone morphogenic protein(BMP) are examples of growth factors that complex to heparin.

The ability of heparin to bind adhesive molecules and growth factors haslead to a number of efforts to use heparin complexes to improveimplantable medical device surfaces by providing surfaces to which cellscan attach and migrate. Other researchers have explored direct coatingsof fibronectin, and peptides and peptide mimetics derived fromfibronectin, with the goal of increasing cell attachment (Walluscheck etal., Eur J Vasc Endovasc Surg, 12:321-30, 1996; Boxus et al., J BioorgMed Chem, 6:1577-95,1998; Tweden et al., J. Heart Valve Dis, 4 Suppl1:S90-7, 1995). Vascular grafts, for example, would be improved by asurface that supports the growth of endothelial cells. Current vasculargrafts of polytetrafluoroethylene and polyethylene terephthalate do notsupport endothelization, and consequently patients must be maintained onlong-term anti-platelet therapy.

Fibronectins function as adhesive, ligand molecules interacting withspecific receptors on the cell surface. Cells types that attach tofibronectin include fibroblasts, endothelial cells, smooth muscle cells,osteoblasts, and chondrocytes.

Other investigators have used heparin/fibronectin complexes to providecell adhesion to polymeric surfaces. For example, heparin-albuminconjugates have been immobilized on carbon dioxide gas plasma-treatedpolystyrene (Bos et al., J. Biomed Mater Res, 47:279-91, 1999) andcomplexed to fibronectin. The fibronectin on these surfaces increasedthe attachment of endothelial cells. Bos et al. (Tissue Eng 4:267-79,1998; J Biomed Mater Res,47:279-91, 1999) reported that endothelialcells grew to confluency on CO₂ gas plasma-treated polystyrene coatedwith an albumin-heparin conjugate. Ishihara et al. (J Biomed Mater Res,50: 144-152, 2000) reported that a heparin-conjugated polystyrenepromoted cell attachment of fibroblasts, smooth muscle cell andendothelial cells. The fibroblasts grown on heparin-conjugatedpolystyrene had growth rates at least comparable to fibronectin-coated,gelatin-coated, or tissue culture-treated media.

A simple method of efficiently complexing fibronectin or other adhesivemolecules, including derivatives or mimics of the foregoing, to aheparin complex would have wide applicability for attaching cells toprostheses, including vascular grafts, bone and cartilage implants,nerve guides and the like. Particularly needed is a method andcomposition permitting use of a wide variety of adhesive molecules,including fibronectin, laminin and the like, as part of a coating forimplantable medical devices. There remains a need in the art for coatingcompositions for implantable medical devices that promote cellularattachment, and further wherein cellular attachment can be modulated bythe quantity of adhesive molecule, and which can be applied simply andeasily with no specialized equipment or techniques.

SUMMARY OF THE INVENTION (DISCLOSURE OF THE INVENTION)

The present invention provides an amphiphatic cell-attachment coatingcomposition for medical device surfaces, which composition includes acovalent complex of from 1 to 30 hydrophobic silyl moieties of FormulaI:

wherein

R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group,

each R₂ is independently selected from the group consisting of C₁₋₁₈alkyl and C₆₋₃₂ aryl

R₃ is N or O, and

n is a number from 1 to 10

directly bound to sodium heparin via covalent bonding, with an adhesivemolecule directly bound to the sodium heparin. The hydrophobic silylmoieties may be bound to the surfaces via hydrophobic bondinginteractions. Further, the complex can include from 2 to 25 hydrophobicsilyl moieties covalently bound to one heparin molecule. In Formula I,R₁ can be benzyl and R₂ can be an alkyl. In a preferred embodiment, thecomplex is [benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate or[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate. In apreferred embodiment, the adhesive molecule is fibronectin; inalternative embodiments, the adhesive molecule may be laminin,vitronectin, thrombospondin, gelatin, polylysine, polyornithine, peptidepolymers containing adhesive sequences and heparin binding sequences,sulfated complex carbohydrates, dextran sulfate, growth hormones,cytokines, lectins, or peptidic polymers thereof.

The invention further provides a non-thrombogenic medical device forcellular attachment, including surfaces for contacting blood, whichsurfaces have coated thereon an non-thrombogenic coating compositioncomprising a covalent complex of from 1 to 30 hydrophobic silyl moietiesof Formula I:

wherein

R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group,

each R₂ is independently selected from the group consisting of C₁₋₁₈alkyl and C₆₋₃₂ aryl,

R₃ is N or O, and

n is a number from 1 to 10

directly bound to heparin via covalent bonding, with an adhesivemolecule directly bound to the heparin. The hydrophobic silyl moietiesmay be bound to the coated surfaces via hydrophobic bondinginteractions. Further, the complex can include from 2 to 25 hydrophobicsilyl moieties covalently bound to one heparin molecule. In Formula I,R₁ can be benzyl and R₂ can be an alkyl. In a preferred embodiment, thecomplex is [benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate or[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate. In apreferred embodiment, the adhesive molecule is fibronectin; inalternative embodiments, the adhesive molecule may be laminin,vitronectin, thrombospondin, gelatin, polylysine, polyornithine, peptidepolymers containing adhesive sequences and heparin binding sequences,sulfated complex carbohydrates, dextran sulfate, growth hormones,cytokines, lectins, or peptidic polymers thereof. The devices of thisinvention include blood gas exchange devices, blood filters, artificialblood vessels, artificial valves, prosthetics, blood shunts, catheters,bone replacements, cartilage replacements and nerve growth guides.

In yet another embodiment, the invention provides a method for renderinga tissue- or blood-contacting surfaces of a medical device resistant tofibrin accumulation while promoting cellular adhesion, which methodincludes coating the surfaces with an non-thrombogenic coatingcomposition comprising a covalent complex of from 1 to 30 hydrophobicsilyl moieties of Formula I:

wherein

R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group,

each R₂ is independently selected from the group consisting of C₁₋₁₈alkyl and C₆₋₃₂aryl,

R₃ is N or O, and

n is a number from 1 to 10

directly bound to heparin via covalent bonding, and attaching to theheparin an adhesive molecule. In this method, the hydrophobic silylmoieties can be bound to the surfaces via hydrophobic bondinginteractions. From 2 to 25 hydrophobic silyl moieties can be covalentlybound to one heparin molecule. In a preferred embodiment, R₁ is benzyland R₂ is an alkyl. In preferred embodiments, the complex is[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate or[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate. The methodcan further include the step of solubilizing the complex in a solventprior to the step of coating the surface. In one embodiment, the step ofcoating the surfaces includes dipping the surface into the coatingcomposition including the complex. In another embodiment, the step ofcoating the surface includes pumping the coating composition includingthe complex onto the surface. In a preferred embodiment of this method,the adhesive molecule is fibronectin; in alternative embodiments, theadhesive molecule may be laminin, vitronectin, thrombospondin, gelatin,polylysine, polyornithine, peptide polymers containing adhesivesequences and heparin binding sequences, sulfated complex carbohydrates,dextran sulfate, growth hormones, cytokines, lectins, or peptidicpolymers thereof. The method can also include the step of solubilizingthe adhesive molecule in a solvent prior to the step of attaching theadhesive molecule. The step of attaching the adhesive molecule includesdipping the surface coated with the complex into a solubilized adhesivemolecule composition. Alternatively, the step of attaching the adhesivemolecule includes pumping the solubilized adhesive molecule compositiononto a surface coated with the complex.

A primary object of the present invention is to provide an amphipathicheparin-fibronectin coating composition for implantable medical devices,which promotes cellular attachment.

A further objective of the invention is to provide a coating thecomposition of which can be varied, such that absent an adhesionmolecule the coating inhibits fibrin deposition, but when the coatingincludes an adhesion molecule, the coating promotes cellular attachmentand cell growth.

A further object of the invention is to provide coating compositionsutilizing fibronectin, derivations of fibronectin, peptide mimics offibronectin, laminin, vitronectin, thrombospondin, gelatin, collagen andsubtypes thereof, gelatin, polylysine, polyornithine, and other adhesivemolecules or derivatives or mimics of other adhesive molecules.

A further object of the present invention is to provide a cost effectiveand commercially feasible method for coating polymeric medical devices,including biodegradable medical devices, with a non-thrombogenic coatingthat inhibits cell attachment.

A further object of the present invention is to provide a cost effectiveand commercially feasible method for coating polymeric medical devices,including biodegradable medical devices, with a coating that resistsfibrin accumulation and promotes cell attachment and growth utilizing acomplex with an adhesive molecule, including fibronectin, peptide mimicsof fibronectin, laminin, vitronectin, thrombospondin, gelatin, collagenand subtypes thereof, gelatin, polylysine, polyornithine, and otheradhesive molecules or derivatives or mimics of other adhesive molecules.

A primary advantage of the present invention is that it provides forcoating medical devices of complex geometries and surfaces with adurable and low-cost coating that promotes uniform cell growth andattachment.

Other objects, advantages and novel features, and further scope ofapplicability of the present invention will be set forth in part in thedetailed description to follow, taken in conjunction with theaccompanying drawings, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instrumentalities andcombinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate several embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating a preferred embodiment of the invention and are not to beconstrued as limiting the invention. In the drawings:

FIG. 1 is a titration plot of the amount of fibronectin andsilyl-heparin needed to support cell attachment; and

FIGS. 2A and B are plots of the absorbance resulting from heparin andfibronectin added in serial doubling dilutions measured using assays.

DESCRIPTION OF THE PREFERRED EMBODIMENTS (BEST MODES FOR CARRYING OUTTHE INVENTION)

The present invention relates to new non-thrombogenic, cell-attachmentcoating compositions. According to the compositions of the presentinvention, hydrophobic moieties are covalently bound to a heparinmolecule to form a covalent, amphipathic complex which may used singlyor in combination with adhesion molecules to promote cell attachment.

As a first aspect, the present invention provides an amphiphatic coatingthat resists fibrin accumulation and inhibits cell attachment. Thecoating comprises a covalent complex of from 1 to 30 hydrophobic silylmoieties of Formula I:

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, and n is a number from 1 to 10, directly boundto a heparin molecule via covalent bonding, with an adhesive moleculecomplexed by affinity interaction to the heparin.

As a second aspect, the present invention provides medical devicescoated with the moieties of Formula I that inhibits fibrin accumulationand cellular adhesion, comprising surfaces for contacting blood andother surfaces where cellular attachment is not desired. Thefibrin-resistant and cellular-adhesion resistant coating compositioncomprises a covalent, amphipathic complex of from 1 to 30 hydrophobicsilyl moieties of Formula I directly bound to a heparin molecule viacovalent bonding but without an adhesive molecule.

As a third aspect, the present invention provides a fibrin-resistantmedical device that promotes surface cellular adhesion, comprisingsurfaces for contacting blood, cells, tissues, or other fluids. Theblood- or cell-contacting surfaces have coated thereon afibrin-resistant and cellular-adhesion coating composition. The fibrinresistant and cellular-adhesion coating composition includes a covalent,amphipathic complex of from 1 to 30 hydrophobic silyl moieties ofFormula I directly bound to a heparin molecule via covalent bonding,wherein an adhesive molecule, including but not limited to fibronectin,is complexed by affinity interaction to the heparin.

As a fourth aspect, the present invention provides a method forrendering blood- and cell-contacting surfaces of a medical deviceresistant to fibrin accumulation while promoting cellular adhesion. Themethod comprises coating the surfaces with an anti-fibrin coatingcomposition. The composition comprises a covalent complex of from 1 to30 hydrophobic silyl moieties of Formula I directly bound to a heparinmolecule via covalent bonding. Thereafter, an adhesion molecule, such asfibronectin, is bound by affinity interaction to the heparin.

As a fifth aspect, the present invention provides a covalent complex ofFormula II:

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, n is a number from 1 to 10, and x is a numberfrom 1 to 30.

These and other aspects of the present invention are described furtherin the description of the preferred embodiment and examples of theinvention which follow.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless otherwise defined, all technical and scientific terms employedherein have their conventional meaning in the art. As used herein, thefollowing terms have the means ascribed to them.

“Alkyl” refers to linear branched or cyclic, saturated or unsaturatedC₁₋₁₈ hydrocarbons such as methyl, ethyl, ethenyl, propyl, propenyl,iso-propyl, butyl, iso-butyl, t-butyl, pentyl, cyclopentyl, hexyl,cyclohexyl, octyl, and the like.

“Aryl” refers to unsaturated C₆₋₃₂ hydrocarbon rings that may besubstituted from 1-5 times with alkyl, halo, or other aryl groups. Arylalso includes bicyclic aryl groups. Specific examples of aryl groupsinclude but are not limited to phenyl, benzyl, dimethyl phenyl, tolyl,methyl benzyl, dimethyl benzyl, trimethyl phenyl, ethyl phenyl, ethylbenzyl, and the like.

“Adhesive molecules” refers molecules which promote cellular attachment,adhesion or growth, including fibronectin, laminin, vitronectin,thrombospondin, heparin-binding domains, and heparan sulfate bindingdomains, as well as synthetic polymers of amino acids containingadhesive sequences derived from any of the foregoing. This includes,without limitation, peptides or polypeptides containing the amino acidswith the single letter codes RGD, IKVAV, YIGSR, and the like. Adhesivemolecules also include lectins that bind to heparin and carbohydratemoieties on the cell surface.

“Heparin” as used herein includes complex carbohydrates or mimetics ofcomplex carbohydrates with properties similar to those of heparin,including heparan sulfate, dextran, dextran sulfate, chondroitinsulfate, dermatan sulfate, and the like.

Herarin Coatina Compositions

The heparin coating compositions of the present invention comprise acovalent complex of one or more hydrophobic silyl moieties with heparin.Heparin is a mixture of variably sulfated polysaccharide chains composedof repeating units of D-glucosamine and either L-iduronic orD-glucuronic acids.

Any suitable form of heparin may be employed in the reaction. Severalsalts of heparin and heparin derivatives are known in the art. Forexample, conventional salts of heparin include sodium heparin, calciumheparin, magnesium heparin, and potassium heparin. Heparin derivativesinclude, but are not limited to ammonium heparin, benzalkonium heparin,and the like. Sodium heparin is one preferred form of heparin forpreparing the covalent complexes according to the present invention. Forthe sake of simplicity, the term “heparin molecule” refers to any ofknown forms of heparin including all salts and derivatives of heparin.

The silyl moiety is represented by the general Formula I:

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, and n is a number from 1 to 10. As will beapparent to those skilled in the art, R₃ is an N or O atom on theheparin molecule, and the unoccupied bond from R₃ signifies theattachment of the silyl moiety to the heparin molecule. Thus, thehydrophobic silyl moiety is capable of attachment to the heparinmolecule at either an O atom of an alcohol (i.e., hydroxyl) or a N atomof an amine.

Heparin comprises many repeating units containing amine and hydroxylfunctional groups which can be the site for attachment of thehydrophobic silyl moiety to the heparin molecule. Accordingly, oneembodiment of the present invention contemplates the attachment of morethan 1 hydrophobic silyl moiety to a single heparin molecule. As many as30 hydrophobic silyl moieties of Formula I or more, and as few as 1hydrophobic silyl moiety may be attached to a single heparin molecule toachieve the covalent complex employed in the heparin coatingcompositions of the present invention. In one embodiment of the presentinvention, between 2 and 25 hydrophobic silyl moieties are attached to asingle heparin molecule. In one embodiment, between 5 and 20 hydrophobicsilyl moieties are attached to a single heparin molecule. In oneembodiment, between 7 and 15 hydrophobic silyl moieties are attached toa single heparin molecule. In one preferred embodiment, 7 or 8hydrophobic silyl moieties are attached to a single heparin molecule. Inanother preferred embodiment 12 hydrophobic silyl moieties are attachedto a single heparin molecule.

In those embodiments wherein more than one hydrophobic silyl moiety isattached to a single heparin molecule, the hydrophobic silyl moietiesmay be attached either through the amine of heparin (e.g., where R₃ isN) or through the hydroxyl group of heparin (e.g., wherein R₃ is O). Inother words, some of they hydrophobic silyl moieties may be attached tothe heparin molecule via bonding at the amine groups of heparin, whileother hydrophobic silyl moieties are attached to the heparin moleculevia bonding at the hydroxyl groups of heparin. It is also possible forall of the hydrophobic silyl moieties to be consistently attached toheparin via one or the other of the amine (e.g., R₃ in all hydrophobicsilyl moieties is N) or the alcohol (e.g., R₃ in all hydrophobic silylmoieties is O).

The bonds between the hydrophobic silyl moieties and the heparinmolecule which effect the attachment of the moieties to the molecule arecovalent bonds. Thus, the coating compositions of the present inventiondo not rely upon ionic interactions between heparin and the hydrophobicmoiety. Rather, the hydrophobic moieties are bonded to the heparinmolecule by covalent bonding through either the amine or hydroxyl groups(or possibly a combination of both amine and hydroxyl groups when two ormore hydrophobic silyl moieties are attached a single heparin molecule).Because the hydrophobic silyl moiety is bound to the heparin moleculethrough covalent bonding, the present invention overcomes one weaknessof conventionally known heparin coatings. Specifically, the problem ofheparin leaching from the coating as a result of the breaking of theionic bond between heparin and the group which attaches heparin to thesurface is overcome by avoiding reliance upon ionic bonding interactionsbetween heparin and the binding group. In the present invention, thecovalent bonds between the hydrophobic silyl moieties and the heparinmolecule in the coating composition are not disrupted by the presence ofionic species in the blood with which the coated surface will come intocontact. The data demonstrate that this process of covalent modificationalso does not lead to detrimental loss of heparin activity as monitoredby a Factor Xa/antithrombin III chromogenic substrate assay on thesurface of target substrates.

The covalent complex according to the present invention can be preparedaccording to the following Scheme 1.

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, n is a number from 1 to 10, and x is a numberfrom 1 to 30.

Generally, the first intermediate, R₁(Si(R₂)₂CH₂)_(n)CI wherein n is 1,is produced by reacting an alkyl or aryl magnesium chloride with achloro(chloromethyl)-dialkyl silane or chloro(chloromethyl)diaryl silanein the presence of tetrahydrofuran (THF). The alkyl or aryl magnesiumchlorides used as starting materials are commercially available, andinclude, for example benzyl magnesium chloride. Thechloro(chloromethyl)dialkyl silane or chloro(chloromethyl)diaryl silanesare also commercially available and include, for examplechloro(chloromethyl)dimethyl silane. The reaction is exothermic, and istypically conducted at temperatures of about 10° C. or less. Thereaction is carried out for a sufficient period of time to yield about80-90% product. Typically the reaction is conducted over a period offrom about 2 to about 24 hours.

First intermediates wherein n is 2 or higher can be produced using aGrignard Reaction involving the reaction of the first intermediatewherein n is 1 with CISi(R₂)₂CH₂CI. This Grignard reaction can berepeated any number of times to achieve the desired value for n in thefirst intermediate. The reaction is carried out in the presence of acatalytic amount of iodine and THF.

The first intermediate (wherein n is 1-10) is converted to the secondintermediate, R₁(Si(R₂)₂CH₂)_(n)OH, by reacting the first intermediatewith potassium acetate (KOAc) in dimethyl formamide (DMF), at atemperature of above about 120° C., and preferably about 135° C. forbetween 12 and 24 hours. The product of this reaction is then reactedwith sodium methoxide (NaOMe) in methanol (MeOH) under reflux for about2 hours to achieve the second intermediate.

The second intermediate is converted to the last intermediate,R₁(Si(R₂)₂CH₂)_(n)OCO₂N(COCH₂)₂, by a two-step reaction process. In thefirst step, the second intermediate is reacted with triphosgene andsodium carbonate in methylene chloride at a temperature of less than 10°C., and preferably about 0° C. The product of this reaction is reactedwith N-hydroxysuccinimide and triethylamine (Et₃N) in methylene chlorideat a temperature of less than 10° C., and preferably about 0° C.

The final intermediate is covalently conjugated to heparin by reactingheparin with the final intermediate in a suitable solvent (e.g.,water/dimethyl formamide) at a pH of about 8.0 to 9.0, and preferablyabout 8.5. The pH of the reaction is controlled by the addition of basesuch as sodium hydroxide, as needed.

Using these general methods, the covalent complexes of the presentinvention can be produced.

The covalent complexes have the general Formula III:

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, n is a number from 1 to 10, and x is a numberfrom 1 to 30.

Preferred complexes include those complexes wherein R₁ of thehydrophobic silyl moiety is aryl. In one preferred embodiment, R₁ isbenzyl. In one preferred embodiment, each R₂ is alkyl. In oneparticularly preferred embodiment, each R₂ is selected from the groupconsisting of methyl, ethyl, propyl, and isopropyl, particularly methyl.In one preferred embodiment, n is a number from 2 to 3.

Specific examples of covalent complexes according to the presentinvention include but are not limited to[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate,[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate, anddodecyl[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.Although these three specific covalent complexes are examples ofcurrently preferred covalent complexes having the general Formula IIabove, other specific examples of such complexes will be apparent tothose skilled in the art and are contemplated by the instant invention.

The silyl-heparin coatings of the present invention comprise thecovalent complexes described above. In addition to the covalent complex,the coating composition may also include one or more solvents whichfacilitate the processes of applying the composition to the surface.Suitable solvents will be those which at least partially solubilize thecovalent complex and which do not interfere with the anti-thrombogenicactivity of heparin. Examples of solvents which may be employed in thecoating compositions of the present invention include but are notlimited to aqueous solvents, alcohols, nitrites, amides, esters,ketones, ethers, and the like. “Aqueous” with reference to solutions orsolvents refers to solutions or solvents which consist primarily ofwater, normally greater than 90 weight percent water, and can beessentially pure water in certain circumstances. For example, an aqueoussolution or solvent can be distilled water, tap water, or the like.However, an aqueous solution or solvent can include water havingsubstances such as pH buffers, pH adjusters, organic and inorganicsalts, alcohols (e.g., ethanol), sugars, amino acids, or surfactantsincorporated therein. The aqueous solution or solvent may also be amixture of water and minor amounts of one or more cosolvents, includingagronomically suitable organic cosolvents, which are miscible therewith,or may form an emulsion therewith. Examples of suitable alcohol solventsinclude but are not limited to methanol, ethanol, propanol, isopropanol,hexanol, as well as glycols such as ethylene glycol, and the like.Examples of suitable nitrites include acetonitrile, propionitrile,butyronitrile, benzonitrile, and the like. Examples of suitable amidesinclude formamide, N,N-dimethylformamide, N,N-dimethylacetamide, and thelike. Examples of suitable esters include methyl acetate, ethyl acetate,and the like. Examples of suitable ketones include acetone, methyl ethylketone, diethyl ketone, and the like. Examples of suitable ethersinclude diethyl ether, tetrahydrofuran, dioxane, dimethoxyethane, andthe like. Any two or more of any of the foregoing solvents may beutilized in the coating composition as well. Currently preferredsolvents include water, particularly distilled water, isopropanol,acetonitrile, and combinations of any two or more of these solvents.

In one preferred embodiment, the covalent complex is solubilized insolvent to achieve a concentration of between about 0.01 and about 10percent by weight, preferably between about 0.1 and about 1 percent, andmore preferably about 0.125 percent.

In addition to the foregoing solvents, the heparin coating compositionsof the present invention may also include therein various conventionaladditives. Examples of additives which may be incorporated into thecompositions of the present invention include but are not limited tobenzalkonium, 4-dimethylaminopyridinium, tetrabutylammonium halides, andthe like.

The coating composition may be coated onto any of a wide variety ofsurface materials to provide anti-thrombogenic effects when the coatedsurface is contacted with blood. Suitable surfaces which may be coatedwith the coating composition of the present invention include anysurface which has an affinity or attraction to the hydrophobic silylmoiety. Such surfaces are typically hydrophobic surfaces. Examples ofsuitable surfaces include but are not limited to hydrophobic polymerssuch as polycarbonate, polyester, polypropylene, polyethylene,polystyrene, polytetrafluoroethylene, polyvinyl chloride, polyamide,polyacrylate, polyurethane, polyvinyl alcohol, polyurethane,polycaprolactone, polylactide, polyglycolide and copolymers of any twoor more of the foregoing; siloxanes such as2,4,6,8-tetramethylcyclotetrasiloxane; natural and artificial rubbers;glass; and metals including stainless steal and graphite.

The heparin coating composition can be applied to the surface to renderthe blood-contacting surface resistant to fibrin accumulation andresistant to cell attachment. Any suitable method for applying thecoating composition to the surface may be employed. One suitable methodfor applying the coating composition to the blood-contacting surface tobe treated is by dipping the blood-contacting surface into the coatingcomposition containing the covalent complex of the present invention. Aliquid coating composition containing the covalent complex of thepresent invention may be prepared using any of the solvents describedabove. The surface is dipped or immersed into a bath of the coatingcomposition. Typically, the dipping process is carried out at elevatedtemperatures, such as between about 30° C. and about 80° C. for a periodof between about 5 and about 20 minutes, preferably about 10 minutes.Thereafter, the surface is allowed to remain in contact with the coatingcomposition containing the covalent complex for a period of betweenabout 15-60 minutes, preferably about 20 minutes, at room temperature.

Another method that may be employed for coating or applying the heparincoating compositions of the present invention onto blood- orcell-contacting surfaces includes a pumping or spraying processes.According to the pumping process, the coating solution having aconcentration of between 0.05 and about 5 percent (w/v) is pumpedthrough the device, where the blood or cell contact will occur, forabout 30 minutes. Thereafter the excess coating materials is washed outwith water or saline. The blood- or cell-contacting surface can becoated by the material of the current invention simply by spraying withthe above-mentioned coating solution as well. The coated surface istypically washed with water before drying.

Following coating of the composition onto the surface, the surface istypically washed with water or saline prior to drying. Advantageously,the foregoing methods for applying the coating composition to a surfaceare relatively quick, commercially feasible and cost-effective. Theyrequire no special equipment or special technical training, and can beapplied to devices with complex surface geometries.

The hydrophobic interactions between the hydrophobic surfaces to becoated and the hydrophobic silyl moieties of the covalent complex formthe bond between the covalent complex and the surface. This hydrophobicinteraction is sufficiently strong so as to provide a stable bondbetween the covalent complex and the surface. The present inventors havenow discovered a method for binding heparin to a surface by usinghydrophobic binding interactions which provide certain advantages overthe method relied upon in previous coating technologies. The presence ofionic species in blood does not disrupt the hydrophobic interactionbetween the covalent complex of the present invention and the surface.

Fibronectin Complexation

Fibronectin has known and demonstrated affinity for heparin.Fibronectins are composed of two similar protein chains, with each chainincluding one domain for cell binding and two domains, one at each endof the chain, for heparin binding. Affinity binding of fibronectinresults in an affinity constant of approximately 10⁸ M.

Any form of fibronectin may be employed, including fibronectin derivedfrom cells, plasma, or tissues, and may be natural or geneticallyengineered, and may be of human origin or derived from another animalspecies. Other adhesive molecules, as defined above, may also beemployed, utilizing the methods described herein for fibronectin.

Following coating of the surface with a silyl-heparin complex, thefibronectin may be affinity complexed to the heparin, resulting in asilyl-heparin-fibronectin complex. To affinity complex the fibronectin,the fibronectin is solubilized in one or more solvents which facilitatethe processes of applying the fibronectin composition to thesilyl-heparin-coated surface. Suitable solvents will be those which atleast partially solubilize fibronectin and which do not interfere withthe activity of heparin or the cellular-attachment activity offibronectin. Examples of solvents that may be employed in the presentinvention include aqueous solutions; aqueous solutions containingalcohols, nitrites, amides, esters, ketones, ethers, and the like; andalcohols, nitrites, amides, esters, ketones, ethers, and the like.Aqueous solutions are thought to be particularly useful fornon-synthetic fibronectins and organic-based solvents for syntheticfibronectins and peptides derived therefrom. “Aqueous” with reference tosolutions or solvents refers to solutions or solvents that consistprimarily of water, normally greater than 90 weight percent water, andcan be essentially pure water in certain circumstances. For example, anaqueous solution or solvent can be distilled water, tap water, or thelike. However, an aqueous solution or solvent can include water havingsubstances such as pH buffers, pH adjusters, organic and inorganicsalts, alcohols (e.g., ethanol), sugars, amino acids, or surfactantsincorporated therein. The aqueous solution or solvent may also be amixture of water and minor amounts of one or more cosolvents, includingagronomically suitable organic cosolvents, which are miscible therewith,or may form an emulsion therewith.

The fibronectin composition can be applied to the silyl-heparin-coatedsurface to render the blood- or cell-contacting surface suitable forcellular adhesion and attachment. Any suitable method for applying thefibronectin composition to the surface may be employed. One suitablemethod for applying the fibronectin composition to thesilyl-heparin-coated surface to be treated is by dipping thesilyl-heparin-coated surface into the fibronectin composition of thepresent invention. The silyl-heparin-coated surface is dipped orimmersed into a bath of the fibronectin composition. Typically, thedipping process is carried out at elevated temperatures, such as betweenabout 30° C. and about 80° C., and preferably between about 40° C. andabout 50° C., for a period of between about 5 and about 60 minutes,preferably between about 20 and about 30 minutes.

Another method that may be employed for coating or applying thefibronectin compositions of the present invention on tosilyl-heparin-coated surfaces includes a pumping or spraying processes.According to the pumping process, the coating solution having aconcentration of between 0.05 and about 5 percent (w/v) is pumpedthrough the device where the blood contact will occur for about 30minutes. Thereafter the excess coating materials is washed out withwater or saline. The blood-contacting surface can be coated by thematerial of the current invention simply by spraying with theabove-mentioned fibronectin solution as well. The coated surface istypically washed with water before drying.

Following attachment of the fibronectin to the heparin of thesilyl-heparin coating of the composition onto the surface, the surfaceis typically washed with water or saline prior to drying.Advantageously, the foregoing methods for applying the coatingcomposition to a surface are relatively quick, commercially feasible andcost-effective.

In an alternative embodiment, the fibronectin may be complexed in anaqueous solution with silyl-heparin, and then the entiresilyl-heparin-fibronectin complex attached to the medical device. Inthis approach, the silyl-heparin is dissolved directly in water andmixed with a predetermined amount of fibronectin such that thefibronectin is essentially entirely complexed with the silyl-heparin. Tothe solution is slowly added an organic solvent such as isopropanol to aconcentration of between 20% and 80% and preferably about 35%. Underthese conditions the silyl-heparin-fibronectin complex undergoes theformation of micelles, with the fibronectin-heparin portion of thecomplex sequestered on the inside of the micelle. A solution of micellescan then be applied to the surface of an appropriate material, therebyallowing the micelles to associate by hydrophobic interaction. Rinsingin an aqueous solution is used to remove excess unbound material andallow the heparin-fibronectin to associate in the aqueous phase. Thisapproach is particularly applicable to fibronectin-derived peptidepolymers.

In yet another embodiment, molecules may be employed in addition to theadhesive molecules defined above. These include, without limitation,polylysine, polyornithine, and similar molecules with net positivecharges that associate with heparin by charge-charge interaction andthereby provide cell adhesive properties. Polylysine and polyornithineare known to enhance cell attachment on certain types of cell cultureware, and may be applied and bound to silyl-heparin complexes asdescribed herein.

A variety of growth factors and cytokines can also be complexed toextracellular matrix heparin and heparan sulfate. The bound growthfactors can thereby be used to promote cell adhesion by providing adisplay of ligands to which cell surface receptors can bind. Fibroblastgrowth factor (FGF), platelet-derived growth factor (PDGF), and bonemorphogenic protein (BMP) are examples of growth factors that complex toheparin. Similarly, cytokines are known to interact with heparin, andcytokines, such as gamma-interferon, may be complexed to extracellularmatrix heparin and heparan sulfate compositions of this invention,including the silyl-heparin substrate.

In yet another embodiment, more than one type of adhesive molecule maybe complexed to silyl-heparin substrate. Thus, both a growth factor andfibronectin may be applied to silyl-heparin such that the final coatingcontains both types of adhesive molecules. The fibronectin is bound byintegrins on the cell surface while a fibroblast growth factor, forexample, is bound by its own distinct set of receptors. The bound growthfactor also provides a vehicle to maximize cellular repopulation of thecoated surface.

The heparin and fibronectin coating compositions of the presentinvention can be applied to the blood-contacting, tissue-containing, orcell contacting surfaces of any of a wide variety of medical devices toprovide the medical device with one or more surfaces promoting cellularadhesion and attachment. Examples of specific medical devices which maybe advantageously treated with the coating compositions of the presentinvention include but are not limited to artificial blood vessels, bloodshunts, nerve-growth guides, artificial heart valves, prosthetics,pacemaker leads, in-dwelling catheters, cardiovascular grafts, bonereplacements, wound healing devices, cartilage replacement devices,urinary tract replacements and the like. Other examples of medicaldevices which would benefit from the application of thecellular-adhesive coating compositions of the present invention will bereadily apparent to those skilled in the art of surgical and medicalprocedures and are therefore contemplated by the instant invention.

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereof. In these examples, “μL”means microliter, “mL” means milliliter; “L” means liter, “μg” meansmicrogram, “mg” means milligram, “g” means gram, “mol” means moles, “M”means molar concentration, “Me” means methyl; “Bn” means benzyl,“nBu₄NI” means tetrabutyl-ammonium iodide, “° C.” means degreesCentigrade. All percentages are in percent by weight unless otherwiseindicated.

EXAMPLE 1 Method for Preparing Silyl-heparin Covalent Complexes.Synthesis of Benzyl(chloromethyl)dimethylsilane

In a 2 L 3-necked flask equipped with a nitrogen inlet, a 500 mldropping funnel and a thermometer, was placed 500 ml of tetrahydrofuran.Chloro(chloromethyl)-dimethylsilane (100 ml, 0.744 mol) was added bysyringe and the colorless solution cooled to 0° C. in an ice/acetonebath. Then benzylmagnesium chloride (2.0 M solution, 400 ml, 0.8 mol)was transferred to the dropping funnel by syringe and added dropwiseover 2 hours. A slightly exothermic reaction was observed and thetemperature was maintained below 10° C. After addition of thebenzylmagnesium chloride was complete, the ice bath was allowed to warmup to room temperature without heating, and the reaction mixture wasstirred overnight. Thereafter hexane (300 ml) was added and the reactionmixture was worked up by dropwise addition of saturated aqueous ammoniumchloride (300 ml) and transferred to a 2 L separatory funnel withadditional hexane (300 ml). After partitioning, the organic layer waswashed with saturated aqueous ammonium chloride (200 ml) and saturatedaqueous sodium chloride (200 ml). The combined aqueous layers werebackwashed with hexane (2×500 ml). The combined organic layers weredried over magnesium sulfate, evaporated on a rotary evaporator, andfinally evaporated with an oil pump to give a colorless oil 162.0 g(109.5% yield). A quantitative yield was assumed with a purity of thecrude product as 91.3%.

Grignard Reaction of Bn(SiMe₂CH₂)_(n)CI and CISiMe₂CH₂CI to giveBn(SiMe₂CH₂)_(n+1)CI

In a 500 ml 3-necked flask equipped with a condenser-nitrogen inlet, aseptum and a thermometer, was placed magnesium powder (7.5 g, 0.31 mol),a catalytic amount of iodine and tetrahydrofuran (100 ml). The brownmixture was heated to reflux briefly with a heat gun until the color ofiodine disappeared. Bn(SiMe₂CH₂)_(n)CI (0.2 mol) was added by syringeand washed down with the tetrahydrofuran (2×25 ml). The reaction wasinitiated with a heat gun. An exothermic reaction was observed and thereaction flask was placed in a water bath until the exothermic reactionsubsided. The resulting grey mixture was heated to reflux for 24 hours.The reagent was then cooled to room temperature and cannulated into apressure filter funnel where it was added directly into another 500 mlround bottom flask in which was placed a solution of CISiMe₂CH₂CI (27.0ml, 0.2 mol) in tetrahydrofuran (50 ml) at room temperature. Themagnesium residue was washed down with tetrahydrofuran (2×25 ml). Thereaction mixture was heated to reflux overnight. The resulting greysuspension was worked up by addition of saturated aqueous sodiumbicarbonate (50 ml) and transferred to a 500 ml separatory funnel withhexane (200 ml). After partition, the organic layer was washed withsaturated aqueous sodium bicarbonate (50 ml) and saturated aqueoussodium chloride (50 ml). Then the combined aqueous layers wereback-washed with hexane (2×100 ml). The combined organic layers weredried over magnesium sulfate, evaporated on a rotary evaporator andfinally evaporated on the oil pump to give an amber oil, which can bepurified by distillation to give a colorless oil. Yield is approximately80 percent.

Conversion of Bn(SiMe₂CH₂)_(n)CI to Bn(SiMe₂CH₂)_(n)OH

Bn(SiMe₂CH₂)_(n)CI (0.16 mol) was dissolved in dimethylformamide (300ml) in a 1 L 3-necked flask. Potassium acetate (50 g, 0.5 mol) was addedfollowed by nBu₄NI (4.0 g, 0.01 mol) and the reaction mixture wasstirred in a 135° C. oil bath for 24 hours. The reaction mixture wasworked up by cooling to room temperature, transferred to a 1 Lseparatory funnel with hexane (500 ml), and washed with saturatedaqueous sodium chloride (3×100 ml). The combined aqueous layers wereback-washed with hexane (3×300 ml). The combined organic layers driedover magnesium sulfate, and evaporated on a rotary evaporator to give anamber oil. The oil was dissolved in methanol (400 ml). Then a generousamount of freshly prepared sodium methoxide was added to adjust the pHto >10 and the reaction mixture was heated to reflux for 2 hours. Thereaction mixture was worked up by neutralizing with acetic acid (AcOH),and evaporated to dryness. The dried mixture was chromatographed withsilica gel in a 6.5×100 cm (height of silica 40 cm) flash column andeluted with 0-30% ethylacetate/hexane to give the desired product as aslightly yellow oil. The yield is approximately 80 percent.

Conversion of Bn(SiMe₂CH₂)_(n)OH to Bn(SiMe₂CH₂)_(n)OCO₂N(COCH₂)₂

Triphosgene (60 g, 0.2 mol) was dissolved in methylene chloride (200 ml)and stirred at 0° C. under nitrogen in a 1 L 3-necked flask equippedwith thermometer, dropping funnel and nitrogen inlet. Sodium carbonate(65 g, 0.6 mol) was added followed by Bn(SiMe₂CH₂)_(n)OH (0.13 moldissolved in 200 ml methylene chloride) dropwise over 30 minutes.Thereafter, the ice/acetone bath was allowed to warm to room temperaturewithout addition of heat. The reaction mixture was allowed to stirovernight and worked up the next morning by filtering through a sinteredglass funnel, which was washed down with toluene (PhCh₃) (200 ml).Thereafter the filtrate was evaporated on a rotary evaporator to give acolorless oil, which was dissolved in methylene chloride and stirred inan ice bath under nitrogen. N-Hydroxysuccinimide (30 g, 0.26 mol) wasadded, followed by dropwise addition of triethylamine (Et₃N) (40 ml,0.28 mol) over 15 minutes. The resulting cloudy mixture was stirred atroom temperature for 1 hour. The reaction mixture was then worked up bydiluting with hexane (600 ml), washed with saturated aqueous ammoniumchloride (3×100 ml), and the combined aqueous phases backwashed withhexane (2×200 ml). The combined organic phases were dried over magnesiumsulfate and evaporated to dryness on a rotary evaporator to give anamber oil. The oil was chromatographed with silica gel in a 6.5×100 cm(height of silica 40 cm) flash column and eluted with 20-50% ethylacetate/hexane to give an amber syrup. The yield is approximately 70percent.

Conjugation of Hepanin with Bn(SiMe₂CH₂)_(n)OCO₂N(COCH₂)₂

Heparin (ammonium free, average molecular weight 10,000; 100 g, 10 mmol)was dissolved in 500 ml of water in a 4000 ml beaker with stirring. DMF(400 ml) was added followed by DMAP (5.0 g, 40 mmol) and the pH wasmonitored by a 702 SM Titrino with program set at: end pont =8.50, maxflow rate =1 ml/min., min. flow rate =10 μl/min., pause time =60 sec.,stop criteria =time (inf.) and connected to a reservoir of 1 M sodiumhydroxide. Bn(SiMe₂CH₂)_(n)OCO₂N(COCH₂)₂ (10×mmol) in DMF (100 ml) wasadded and the pH began to drop. The program was started as soon as thepH dropped to just below 8.5. The resulting milky mixture was allowed tostir at room temperature while the pH of the reaction mixture wasmaintained at 8.5 by Titrino by automatic addition of 1 M sodiumhydroxide as necessary. The amount of 1 M sodium hydroxide used (in ml)was plotted against reaction time (in hours) as the reaction profile.The reaction mixture was worked up, when the reaction profile begins toflatten out, by trituration with acetone (2 I) and the white suspensionis filtered through a sintered glass funnel to give a white solidresidue, which was contaminated with DMF and some of the residualN-hydroxy-succinimide. This crude material can be purified by soxhletextraction with acetone overnight to give a white powder. The yields aregenerally greater than 95%.

Procedure for Coating the Complexes onto the Surface

The complex is completely dissolved in ⅓ volume of distilled water withgentle stirring. A solvent such as isopropyl alcohol or acetonitrile isadded in the amount of ⅔ volume and the solution is mixed. The thusprepared coating solution has a complex concentration of between 0.01and 10 percent based upon the weight of the solution. The material to becoated is dipped in the coating solution at elevated temperaturesusually ranging from 30° C. to 50° C. for about 10 minutes, followed bystanding in room temperature for about 20 minutes. The coated materialis taken out of the coating solution and rinsed thoroughly withdistilled water or saline solution prior to drying.

EXAMPLE 2 One Technique for Applying Silyl-Heparin Coating Compositionsto Surfaces

The covalent complex (100 mg) produced according to Example 1 above issolubilized in ⅓ volume, 27 ml of distilled water with gentle stirring.Thereafter ⅔ volume, 53 ml of isopropyl alcohol or acetonitrile isadded. The resulting concentration of the covalent complex in solutionis about 0.125 percent by weight. The blood-contacting surface to becoated with the coating composition is dipped into the coatingcomposition for 10 minutes at a temperature of between 30° C. to 50° C.Thereafter, the surface is allow to remain in contact with the coatingcomposition for approximately 20 minutes, at room temperature.Thereafter, the coated surface is removed from the coating compositionand rinsed thoroughly with distilled water or saline solution.

EXAMPLE 3 Stability of Heparin Coating Compositions on Surfaces Exposedto Ionic Environments

Various surfaces coated according to Example 2 were evaluated forheparin activity after washing with 3 percent (by weight) sodiumchloride solution. Surface heparin activity is measured in mIU/cm²according to the technique described in Sigma Diagnostics, Heparin,Procedure No. CRS 106.

Results obtained from the evaluation of average heparin activity onvarious surfaces after washing with sodium chloride are set forth inTable 1 below. The concentration of the covalent complex in the coatingsolution was 0.25% (W/V).

TABLE 1 Percent by volume of isopropyl alcohol in IPA/H₂O solventMaterial Coated 50 55 60 65 70 75 POLYCARBONATE 6.8 8.0 17.6 16.3 14.714.9 TMCTS 0.4 3.8 3.5 2.0 1.1 2.4 POLYESTER 4.5 4.4 5.5 3.7 5.3POLYVINYL CHLORIDE 2.6 2.9 10.0 6.5 4.0 2.8 STAINLESS STEEL 12.9 13.18.3 11.0 12.9 13.8

EXAMPLE 4 Silyl-Heparin Application Prior to Fibronectin Attachment

Benzyl magnesium chloride was treated serially with chloro(chloromethyl)dimethylsilane to give a benzyl-(1,2 dimethyl)disilyl compound. Thebenzyldisilyl compound was modified to form an activated succinimidylester that was, in turn, conjugated to heparin to form a benzyl-(1,2dimethyl)disilyl heparin. This is shown in Scheme 2 below.

The silyl-heparin was used as a 1% solution in 70% acidified, aqueousethanol. To coat wells, the silyl-heparin solution was applied in 20μL/well for 15 minutes at 50-60° C. The wells were then rinsed severaltimes in saline and air-dried. To coat other compositions, thecomposition were immersed in the 1% silyl-heparin solution as describedfor 15 minutes at 50-60° C., and rinsed extensively in water or saline.

The silyl-heparin coating may be applied to any polymeric substrate,either forming a medical or other implantable device, or coated orotherwise forming a surface of a medical or other implantable device.The coating was applied, as described, to polystyrene and polyurethanepolymeric surfaces. The polymeric substrate includes biodegradablepolymers, including, for example, polylactide, polylactide:polyglycolideand polycaprolactone, to which the silyl-heparin coating was applied.The silyl-heparin coating may also be applied to any metallic substrate,and was applied to stainless steel.

EXAMPLE 5 Attachment of Fibronectin to Silyl-Heparin Coated Substrate

Fibronectin was attached to the silyl-heparin coated substrates ofExample 4 by incubation of in an aqueous 0.9% saline solution and 20μg/mL bovine plasma fibronectin. After 30 minutes the unboundfibronectin was rinsed off. The silyl-heparin-fibronectin coatedsubstrates were then either used directly or air dried and stored forsubsequent use.

EXAMPLE 6 Detection of Heparin and Fibronectin

Using surfaces coated with silyl-heparin as in Example 4, and to whichfibronectin was attached as in Example 5, the presence of both heparinand fibronectin was detected in analytical assays. Heparin was detectedusing a commerically-available an enzyme-linked assay kit (SigmaChemical Co., St. Louis) that measures the heparin-induced inhibition ofantithrombin/factor Xa as measured with a factor Xa specific chromogenicsubstrate. The enzyme-inhibition assay was performed in low-attachment96 well plates following the directions of the manufacturer.

To insure that the amphipathic heparin did not introduce an unknownvariable in the enzyme inhibition assay, a second assay was performedusing a hydrazine-activated biotin (Yu and Toole, 1995). These assayswere performed in low-attachment 96 well plates. A solution ofhydrazine-activated biotin was added to wells that were uncoated orcoated with silyl-heparin. The solution was composed of 0.1 M sodiumacetate, pH 5.2, containing 350 g of EZ-link biotin-LC-hydrazine (PierceChemical Co.). After 30 minutes, the wells were rinsed in water andquenched in 5% dextrose. A solution of PBS containing 20% serum and a1:1000 dilution horseradish peroxidase-conjugated avidin (HRPO-avidin)was added. After 30 minutes the unbound material was rinse off and achromogenic solution of ABTS (1-Step ABTS, Pierce Chemical Co.) added.Upon color development, an aliquot of 0.2 M sulfuric acid was added tostop the reaction, and the absorbance detected at 650 nm.

Fibronectin was detected immunochemically. The assays were performed inlow-attachment 96 well plates in wells with no coating, a coating offibronectin, or a coating of silyl-heparin/fibronectin. PBS containing asaturating amount of gelatin and rabbit anti-human fibronectin (known tobe cross-reactive with bovine fibronectin) was added to the wells. After30 minutes the primary antibody was removed, the plate rinsed, and asolution of PBS containing a saturating amount of gelatin and goatanti-rabbit IgG was added. After 30 minutes the unbound material wasrinsed off and a chromogenic solution of ABTS (1-Step ABTS, PierceChemical Co.) added. Upon color development, an aliquot of 0.2 Msulfuric acid was added to stop the reaction, and the absorbancedetected at 650 nm.

EXAMPLE 7 Cells Used for Adhesion Studies

Several cell types were used, including GS-9L cells (rat gliosarcoma),C3H10T1/2 (murine fibroblasts), Jurkat (human T cell), bovine aortaendothelial (BAE) cells and rat lymphocytes. GS-9L and C3H100T1/2 weremaintained in log phase growth and detached from cultureware usingVersene and collected by centrifugation. Jurkat cells grew as singlecell suspensions and were collected by centrifugation. Rat lymphocyteswere collected from heparinized blood by density gradient isolation overFicoll-Hypaque® media. The collected cells were rinsed once by low speedcentrifugation and suspended in either serum-free Dulbecco's ModifiedEagles Medium (DMEM) containing pyruvate or RPMI 1640 containing 10%fetal bovine serum supplemented with pyruvate. Aliquots of 10⁴ cells in100 μL were used in subsequent adhesion studies.

The cells were examined after plating using an inverted, phase-contrastmicroscope. At selected time points up to 4 days after initial seeding,the cells were rinsed three times in saline and fixed in buffered 10%formalin or 35% ethanol. In some cases, the cells were stained in situwith a 0.01% aqueous solution of crystal violet. The amount ofattachment was scored visually or quantitated. To establish the relativenumber of cells bound, crystal violet stained cells were dissolved in asolution of 70% ethanol containing 0.1% sodium dodecyl sulfate and 0.38M Tris and the absorbance monitored at 610 nm, using the methods ofScragg and Ferreira (Anal Biochem 198:80-5, 1991) and Grando et al.(SkinPharmacol 6:135-47, 1993).

EXAMPLE 8 Attachment of Cells

The cells of Example 7 were added to untreated polystyrene wells, wellscoated with silyl-heparin as in Example 4, withsilyl-heparin-fibronectin as in Example 5, and with only fibronectin byincubation as described in Example 5. As shown in Table 2, C3H10T1/2fibroblasts, GS-9L gliosarcoma, Jurakt T cells, BAE cells and ratlymphocytes cultured in serum-containing medium on low-attachment,nominally non-adherent polystyrene did not attach to the substrate.Similarly, these cells cultured on silyl-heparin alone did not attach.When seeded following pre-treatment with bovine fibronectin, isolatedC3H10T1/2 cells and isolated GS-9L cells were found attached. When cellswere cultured on silyl-heparin-fibronectin, CH310T1/2, BAE and GS-9Lcells rapidly attached and spread onto the substrate. Cells were seededat a concentration of 10⁴ cells/well in serum-containing medium.

TABLE 2 CELL ATTACHMENT SILYL- SILYL- FIBRO- HEPARIN- CELL TYPEUNTREATED HEPARIN NECTIN FIBRONECTIN GS-9L − − +/− ++++ C3H10T1/2 − −+/− ++++ BAE − +/− − +++ Jurkat − − − − Lympho- − − − +/− cytes

EXAMPLE 9 Adherence and Cell Spread

Using the methods as described in Example 8, GS-9L, C3H10T1/2 and BAEcells were adhered by 1 hour after seeding on silyl-heparin-fibronectin,and many of the cells demonstrated evidence of spreading onto thesubstrate. By 2 hours nearly all of the cells evidenced spreading. Themorphology of GS-9L, C3H10T1/2 and BAE cells after 24 hours onsilyl-heparin-fibronectin were similar to cells grown on conventionaltissue cultureware. The cells grew to confluency by 4 days. Cells seededin serum-free medium attached and spread although not as well as inserum-containing medium, and further did not grow. No effort was made togrow the cells in a defined- or serum-low medium known to supportgrowth. Addition of up to 10 U of heparin to the medium of cells platedonto silyl-heparin-fibronectin coated plates did not inhibit theattachment or growth of C3H10T1/2. The results for C3H10T1/2 cells areshown in Table 3.

TABLE 3 CHARACTERISTIC ATTACHMENT SPREADING GROWTH GS-9L CELLS Serum++++ ++++ ++++ Serum-free +++ +++ − C3H10T1/2 CELLS Serum ++++ ++++ ++++Serum-free +++ +++ −

EXAMPLE 10 Use on Variety of Substrates

The general applicability of silyl-heparin/fibronectin was evaluated byapplying this coating using the methods of Examples 4 and 5 to a varietyof surfaces, including polystyrene, polylactide:polyglycolide,polycaprolactone, polurethane, and stainless steel. As in Example 9above, C3H10T1/2 cells did not attach to polystyrene. C3H10T1/2 cellsalso did not attach to polyurethane. They did, however, bind moderatelywell to polylactide:polyglycolide and to polycaprolactone, and very wellto stainless steel. Regardless of the substrate, coating withsilyl-heparin essentially eliminated cell attachment. The inhibition ofcell attachment was noted even on surfaces known to support cell growth,such as stainless steel. Even when the cells were culture for up to 4days, no cell attachment was evident. Treating the plates with heparinalone did not inhibit attachment. Fibronectin alone did notsignificantly support cell attachment to polystyrene. It did, however,improve the attachment of cells to polyurethane andpolylactide:polyglycolide, and to a lesser extent polycaprolactone.Silyl-heparin-fibronectin increased the cell density for all substratesrelative to the uncoated surfaces, and increased the cell density onpolycaprolactone by about 2 fold. The results are summarized in Table 4.

TABLE 4 C3H10T1/2 CELL ATTACHMENT SILYL- HEPARIN- UN- SILYL- FIBRO-FIBRO- SURFACE TREATED HEPARIN NECTIN NECTIN Polystyrene − − −/+ ++++Polyurethane − − ++++ ++++ Polyactide: ++ − ++++ ++++ polyglycolideStainless steel* ++++ − ++++ ++++ Polycaprolactone* +/− +/− ++ ++++*determined by crystal violet staining

EXAMPLE 11 Determination of Optimal Concentrations of Fibronectin andSilyl-Heparin

The optimal amount of fibronectin and s-heparin to support attachment ofC3H10T1/2 cells was determined by cross-titration, as shown on FIG. 1.The EC₅₀ for fibronectin was approximately 5 μg/well and that ofs-heparin about 8.7 μg/well. The cell attachment curve for thefibronectin dilution had a rapid fall-off, suggestive of a thresholdeffect. The fall-off for the silyl-heparin was more gradual. The curveof the relative amount of silyl-heparin on the substrate followingserial dilution generally followed the cell attachment curve. The curveof the relative amount of fibronectin bound to silyl-heparin did notmirror the cell attachment curve.

In FIG. 1, fibronectin (Δ - - - Δ) or silyl-heparin (• - - - •) weretitrated to determined the quantity needed to support attachment ofC3H10T1/2 cells. All dilutions were performed in quadruplicate in wellsof 96-well plates of low-attachment polystyrene. All dilutions wereperformed in quadruplicate in wells of 96-well plates of low-attachmentpolystyrene. For titration of fibronectin, silyl-heparin was applied at30 μg/well in 70% ethanol as in Example 4. After rinsing, fibronectinwas applied in doubling dilutions of phosphate buffered saline startingfrom 4 μg/well. For titration of silyl-heparin, silyl-heparin wasapplied in doubling dilutions in 70% ethanol starting from silyl-heparinconcentrations of 30 μg/well. Fibronectin was then applied in saline at4 μg/well. Cells were at a concentration of 10⁴ cells/well in growthmedium. For both experiments, the cells were allowed 24 hours to attachand then were rinsed and fixed in buffered formalin. The cells werestained for 5 minutes with 0.01% aqueous crystal violet and theabsorbance at 610 nm determined.

FIG. 2 compares the relative amounts of bound heparin and fibronectin indoubling dilutions to attached cell density. In FIG. 2 A the relativeamount of bound heparin was compared to subsequent attached cell densityfollowing complexation with fibronectin (4 μg/well). In FIG. 2B therelative amount of bound fibronectin (FN) to wells pre-coated withsilyl-heparin (300 μg/well) was compared to subsequent cell densityobtained after fibronectin complexation. Cell density was expressed asthe percent maximal absorbance of crystal violet. Heparin was detectedusing an enzyme-linked assay while fibronectin was detectedimmunochemically.

EXAMPLE 12 Use of Other Adhesive Molecules

Laminin was attached to the silyl-heparin coated substrates of Example 4by incubation in an aqueous 0.9% saline solution containing 20 μg/mLintact murine laminin. After 30 minutes the unbound laminin was removedby rinsing. In studies similar to those in Example 8, C3H10T1/2 cellsattached to surfaces coated with the silyl-heparin-laminin complex.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverin the appended claims all such modifications and equivalents. Theentire disclosures of all references, applications, patents, andpublications cited above are hereby incorporated by reference.

What is claimed is:
 1. An amphiphatic cell-attachment coatingcomposition for medical device surfaces, said composition comprising acovalent complex of from 1 to 30 hydrophobic silyl moieties of FormulaI:

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl R₃ is N or O, and n is a number from 1 to 10 directly boundto sodium heparin via covalent bonding, with an adhesive moleculedirectly bound to the sodium heparin.
 2. The composition according toclaim 1, wherein said hydrophobic silyl moieties bind to said surfacesvia hydrophobic bonding interactions.
 3. The composition according toclaim 1, wherein said complex comprises from 2 to 25 hydrophobic silylmoieties covalently bound to one heparin molecule.
 4. The compositionaccording to claim 1, wherein R₁ is benzyl in said hydrophobic silylmoiety of Formula I.
 5. The composition according to claim 1, whereineach R₂ is an alkyl in said hydrophobic silyl moiety of Formula I. 6.The composition according to claim 1, wherein n is 2 or 3 in saidhydrophobic silyl moiety of Formula I.
 7. The composition according toclaim 1, wherein said complex is[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.
 8. Thecomposition according to claim 1, wherein said complex is[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.
 9. Thecomposition according to claim 1, wherein said adhesive molecule isfibronectin.
 10. The composition according to claim 1, wherein saidadhesive molecule is selected from the group consisting of fibronectin,laminin, vitronectin, thrombospondin, gelatin, polylysine,polyornithine, peptide polymers containing adhesive sequences andheparin binding sequences, sulfated complex carbohydrates, dextransulfate, growth hormones, cytokines, lectins, and peptidic polymersthereof.
 11. A non-thrombogenic medical device for cellular attachment,comprising surfaces for contacting blood, said surfaces having coatedthereon an non-thrombogenic coating composition comprising a covalentcomplex of from 1 to 30 hydrophobic silyl moieties of Formula I:

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, and n is a number from 1 to 10 directly boundto heparin via covalent bonding, with an adhesive molecule directlybound to the heparin.
 12. The device according to claim 11, wherein saidhydrophobic silyl moieties bind to said surfaces via hydrophobic bondinginteractions.
 13. The device according to claim 11, wherein said complexcomprises from 2 to 25 hydrophobic silyl moieties covalently bound toone heparin molecule.
 14. The device according to claim 11, wherein R₁is benzyl in said hydrophobic silyl moiety of Formula I.
 15. The deviceaccording to claim 11, wherein each R₂ is an alkyl in said hydrophobicsilyl moiety of Formula I.
 16. The device according to claim 11, whereinn is 2 or 3 in said hydrophobic silyl moiety of Formula I.
 17. Thedevice according to claim 11, wherein said complex is[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.
 18. Thedevice according to claim 11, wherein said complex is[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.
 19. Thedevice according to claim 11, wherein said device is selected from thegroup consisting of blood gas exchange devices, blood filters,artificial blood vessels, artificial valves, prosthetics, blood shunts,catheters, bone replacements, cartilage replacements and nerve growthguides.
 20. The device according to claim 11, wherein said adhesivemolecule is fibronectin.
 21. The device according to claim 11, whereinsaid adhesive molecule is selected from the group consisting offibronectin, laminin, vitronectin, thrombospondin, gelatin, polylysine,polyornithine, peptide polymers containing adhesive sequences andheparin binding sequences, sulfated complex carbohydrates, dextransulfate, growth hormones, cytokines, lectins, and peptidic polymersthereof.
 22. A method for rendering a tissue- or blood-contactingsurfaces of a medical device resistant to fibrin accumulation whilepromoting cellular adhesion, said method comprising coating saidsurfaces with an non-thrombogenic coating composition comprising acovalent complex of from 1 to 30 hydrophobic silyl moieties of FormulaI:

wherein R₁ is an C₁₋₁₈ alkyl or C₆₋₃₂ aryl group, each R₂ isindependently selected from the group consisting of C₁₋₁₈ alkyl andC₆₋₃₂ aryl, R₃ is N or O, and n is a number from 1 to 10 directly boundto heparin via covalent bonding, and attaching to the heparin anadhesive molecule.
 23. The method according to claim 22, wherein saidhydrophobic silyl moieties bind to said surfaces via hydrophobic bondinginteractions.
 24. The method according to claim 22, wherein said complexcomprises from 2 to 25 hydrophobic silyl moieties covalently bound toone heparin molecule.
 25. The method according to claim 22, wherein R₁is benzyl in said hydrophobic silyl moiety of Formula I.
 26. The methodaccording to claim 22, wherein each R₂ is an alkyl in said hydrophobicsilyl moiety of Formula I.
 27. The method according to claim 22, whereinn is 2 or 3 in said hydrophobic silyl moiety of Formula I.
 28. Themethod according to claim 22, wherein said complex is[benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.
 29. Themethod according to claim 22, wherein said complex is[benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate.
 30. Themethod according to claim 22, wherein further comprising the step ofsolubilizing said complex in a solvent prior to said step of coatingsaid surface.
 31. The method according to claim 22, wherein said step ofcoating said surface comprises dipping said surface into said coatingcomposition comprising said complex.
 32. The method according to claim22, wherein said step of coating said surface comprises pumping saidcoating composition comprising said complex onto said surface.
 33. Themethod according to claim 22, wherein said adhesive molecule isfibronectin.
 34. The method according to claim 22, w wherein saidadhesive molecule is selected from the group consisting of fibronectin,laminin, vitronectin, thrombospondin, gelatin, polylysine,polyornithine, peptide polymers containing adhesive sequences andheparin binding sequences, sulfated complex carbohydrates, dextransulfate, growth hormones, lectins, and peptidic polymers thereof. 35.The method according to claim 22, wherein further comprising the step ofsolubilizing said adhesive molecule in a solvent prior to said step ofattaching said adhesive molecule.
 36. The method according to claim 35,wherein said step of attaching said adhesive molecule comprises dippingsaid surface coated with said complex into said solubilized adhesivemolecule composition.
 37. The method according to claim 35, wherein saidstep of attaching said adhesive molecule comprises pumping saidsolubilized adhesive molecule composition onto said surface coated withsaid complex.